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Abstract: Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.

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Abstract: In this work, we address the kinetics of dehydrogenation occurring at high temperatures (HT) in riebeckite, a sodic amphibole with the ideal composition Na2Fe3+2 Fe2+3Si8O22(OH)2. We did isothermal experiments on both powders and single-crystals up to 560 °C and monitored the O-H stretching signal by Fourier Transform Infrared (FTIR) spectroscopy. Single-crystals show an initial increase in IR absorption intensity due to increasing vibrational amplitudes of the O-H bond stretching, not observed for powders. The OH-intensities vs. time were fitted using the formalism for first-order reactions. The calculated activation energies for H+ diffusion in riebeckite are 159 ± 15 kJ/mol for powders and 216 ± 20 kJ/mol for single crystals, respectively. The exponential factor m in the Avrami-Erofeev equation obtained for crystals ranges between 1.02 and 1.31, suggesting that, unlike powders, the dehydration process in crystals is not a purely first-order reaction. This implies that a second energy barrier must be considered, i.e., diffusion of H+ through the crystal. FTIR imaging showed that H+ diffusion occurs mainly perpendicular to the silicate double-chain. Our results confirm that the release of H+ from riebeckite occurs after the irreversible Fe2+-to-Fe3+ exchange, thus at temperatures > 550 °C. To be effective, the process needs the presence of external oxygen that, by interacting with H+ at the crystal surface, triggers the release of H2O molecules. This implies that oxidizing conditions are required for the amphibole to be an efficient water source at depth.

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Abstract: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are found in a high proportion of diffuse gliomas. The presence of the IDH1 mutation is a valuable diagnostic, prognostic and predictive biomarker for the management of patients with glial tumours. Techniques involving vibrational spectroscopy, e.g., Fourier transform infrared (FTIR) spectroscopy, have previously demonstrated analytical capabilities for cancer detection, and have the potential to contribute to diagnostics. The implementation of FTIR microspectroscopy during surgical biopsy could present a fast, label-free method for molecular genetic classification. For example, the rapid determination of IDH1 status in a patient with a glioma diagnosis could inform intra-operative decision-making between alternative surgical strategies. In this study, we utilized synchrotron-based FTIR microanalysis to probe tissue microarray sections from 79 glioma patients, and distinguished the positive class (IDH1-mutated) from the IDH1-wildtype glioma, with a sensitivity and specificity of 82.4% and 83.4%, respectively. We also examined the ability of attenuated total reflection (ATR)-FTIR spectroscopy in detecting the biomolecular events and global epigenetic and metabolic changes associated with mutations in the IDH1 enzyme, in blood serum samples collected from an additional 72 brain tumour patients. Centrifugal filtration enhanced the diagnostic ability of the classification models, with balanced accuracies up to ~69%. Identification of the molecular status from blood serum prior to biopsy could further direct some patients to alternative treatment strategies.

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Abstract: The demand for xylenes is projected to increase over the coming decades. The separation of xylene isomers, particularly p- and m-xylenes, is vital for the production of numerous polymers and materials. However, current state-of-the-art separation is based upon fractional crystallisation at 220 K which is highly energy intensive. Here, we report the discrimination of xylene isomers via refinement of the pore size in a series of porous metal–organic frameworks, MFM-300, at sub-angstrom precision leading to the optimal kinetic separation of all three xylene isomers at room temperature. The exceptional performance of MFM-300 for xylene separation is confirmed by dynamic ternary breakthrough experiments. In-depth structural and vibrational investigations using synchrotron X-ray diffraction and terahertz spectroscopy define the underlying host–guest interactions that give rise to the observed selectivity (p-xylene < o-xylene < m-xylene) and separation factors of 4.6–18 for p- and m-xylenes.

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Abstract: Vibrational microspectroscopy via Fourier transform infrared (FTIR) faces an experimental trade-off among the signal to noise ratio (SNR), acquisition time, spatial resolution, and sample coverage. This is mainly associated with broadband source type: e.g. low brightness thermal sources with high flux for large field of view imaging at low resolution, or low ´etendue of synchrotron radiation infrared (SRIR) for diffraction-limited scanning mi- croanalysis at high magnification.1 Adaptive optics (AO), in this case deformable mirror (DM), is a potent tool in tackling the problem by modulating the intensity of high brightness structured SRIR beam toward a homo- geneous field illumination for IR imaging at high magnification. The latter is required for an efficient coupling of SRIR source to a multi-pixel detector such as focal plane array (FPA).2 Additionally, DM enables to achieve different shapes, optimized for different Cassegrain IR objective. Regardless, the quality of the generated beam relies upon the performance of the adaptive elements, i.e. actuators and their linear and reproducible response to the applied voltage. Moreover, the beam shaping capability of a single DM in controlling light beam position and angle is limited by its actuators influence function. In this work, we implemented two DMs for intensity shaping for the complex SRIR beam. A variation of multi-conjugate AO is implemented to characterize the performance of DMs and their actuators transfer function at multiple locations. An IR sensitive microbolometer array has been optically conjugated to the focal plane of individual actuators and the far-field of DM, in order to probe the corresponding actuating response. By analysing each actuator’s response individually, a measure of linear independence, uniformity in response, and cross-coupling can be obtained in a spectral range, from visible to near and mid IR. Additionally, by assembling the vectorized version of each actuator response, the transfer matrix can be formed. This matrix describes the relationship between the actuation effect on the beam and the response of the IR microbolometer, at the given conjugate planes. Based on such discussion, we assess the stability of the deformable mirror for open-loop (i.e. without feedback) operation.